The current diphtheria epidemic in the former Soviet Union has resulted in a heightened interest in the bacterium Corynebacterium diphtheriae, the causative agent of diphtheria. With the exception of diphtheria toxin, little information is available concerning the pathogenesis of this organism with respect to its ability to invade, colonize and survive within the human host. The acquisition of iron is an important virulence determinant for many bacterial pathogens, and numerous virulence factors including diphtheria toxin are regulated by iron. The hmuO gene is required for the utilization of heme and hemoglobin as iron sources by C. diphtheriae. The product of hmuO has homology to eukaryotic heme oxygenases which are involved in the degradation of heme and release of iron. Current studies have focused on examining the transcription of the hmuO gene. To investigate the mechanism of hmuO regulation, a promoterless lacZ gene present on the promoter-probe vector pCM502 was placed under transcriptional control of the hmuO promoter. In the C. diphtheriae strain C7, optimal expression from the hmuO promoter was obtained only in the presence of heme or hemoglobin. Expression of hmuO in high iron medium containing heme was repressed 5-6 fold, suggesting that the hmuO promoter was under dual regulation, in which transcription was activated in the presence of heme or hemoglobin and repressed by iron. Gel mobility shift assays and Dnase I footprinting experiments indicated that DtxR binds in a metal-dependent manner to a sequence that overlaps the putative hmuO promoter. Northern blot analysis indicated that the hmuO mRNA was monocistronic and that transcription was heme inducible.